Colorectal cancer (CRC) is one of the most common causes of cancer-related deaths in the world. In 2009, there were more than 146,000 new cases of colorectal cancer diagnosed, and colorectal cancer was the cause of nearly one out of every five cancer-related deaths in the United States. Jemal et al., CA Cancer J Clin, 59:225-49 (2009). White light colonoscopy is currently the primary method for performing colorectal cancer screening. This technique is sensitive to morphological changes in the mucosa, such as polyps and masses. However, this method is not sensitive to the detection of flat and depressed lesions, and the polyp miss rates can be as high as 22%. van Rijn et al., Am J Gastroenterol. 101:343-50 (2006). Adenomatous polyps, or adenomas, appear to be major precursors to CRC, even though only a fraction of adenomas progress to CRC. The transformation from pre-malignant mucosa to carcinoma occurs over a period of many years, thus providing window of opportunity for early detection. Furthermore, the presence of flat dysplastic lesions in the setting of chronic ulcerative colitis presents a significantly increased risk for the development of frank carcinoma.
Pre-clinical mouse models of disease provide an important tool for studying mechanisms of disease development. It has been established that mutations in the adenomatous polyposis coli (APC) gene are likely to be critical events in the initiation of the majority of adenomas and CRC. Previously-reported genetically engineered mouse models that mimic human APC gene mutations mainly develop adenomas in the small intestine (e.g., APCMin model, Su et al., Science 256(5057):668-670 (1992)), not the distal colon, making it difficult to image the polyps and their progression in vivo using currently available small animal endoscopy tools. Hinoi et al., Cancer Res., 67(20): 9721-9730 (2007) describes genetically engineered mice (termed CPC:Apc mice) in which a somatic mutation in an Apc allele leads to a truncated Apc protein and causes the development of adenomas in the distal colon as early as 10 weeks. Others have developed mouse models that grow tumors in the distal colon using implantation of cancerous cells [Alencar et al., Radiology, 244: 232-238 (2007)] or adenovirus activated mutations [Hung et al., Proc. Natl. Acad. Sci. USA, 107: 1565-1570 (2010)] and report binding of cathepsin B smart probes, but surgical intervention was needed to generate polyps and the ensuing response to injury may have resulted in target alteration.
Endoscopic imaging with use of exogenous fluorescent-labeled probes, is a promising method for achieving greater specificity in the detection of neoplastic lesions by identifying the expression of unique molecular targets. Imaging provides precise localization, and fluorescence provides improved contrast. Previously, several diagnostic molecules have been used as targeted agents, including antibodies and antibody fragments, for the detection of pre-malignant and malignant lesions in various types of cancer. However, the use of antibodies and antibody fragments is limited by immunogenicity, cost of production and long plasma half-life. Small molecules, RNA aptamers, and activatable probes have also been used. Peptides represent a new class of imaging agent that is compatible with clinical use in the digestive tract, in particular with topical administration.
Phage display is a powerful combinatorial technique for peptide discovery that uses methods of recombinant DNA technology to generate a complex library of peptides, often expressing up to 107-109 unique sequences, that can bind to cell surface antigens. The DNA of candidate phages can be recovered and sequenced, elucidating positive binding peptides that can then be synthetically fabricated. Phage display identified peptide binders to high grade dysplasia in Barrett's esophagus [Li et al., Gastroenterology, 139:1472-80 (2010)] and human colonic dysplasia [Hsiung et al., Nat. Med., 14: 454-458 (2008)] using the commercially available NEB M13 phage system. The T7 system has proven effective in in vivo panning experiments identifying peptides specific to pancreatic islet vasculature [Joyce et al., Cancer Cell, 4: 393-403 (2003)], breast vasculature [Essler and Ruoslahti, Proc. Natl. Acad. Sci. USA, 99: 2252-2257 (2002)], bladder tumor cells [Lee et al., Mol. Cancer. Res., 5(1): 11-19 (2007)], and liver tissue [Ludtke et al., Drug Deliv., 14: 357-369 (2007)]. Panning with intact tissue presents additional relevant cell targets while accounting for subtle features in the tissue microenvironment that may affect binding.
Improved animal models for CRC and new products and methods for early detection of dysplasia are needed in the art. New products and methods for early detection would have important clinical applications for increasing the survival rate for CRC and reducing the healthcare costs.